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Anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody, nucleocapsid protein technology, applied in antiviral immunoglobulin, antibody, immunoglobulin and other directions, can solve the problem of lack of qualified antibody raw materials, achieve short detection cycle, less workload, high throughput Effect

Pending Publication Date: 2021-12-21
NANJING GENSCRIPT BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are currently no diagnostic products for SARS-CoV-2 virus antigen detection on the market. The main reason is that SARS-CoV-2 virus is a new type of virus, and there is no qualified antibody raw material for the development of SARS-CoV-2 virus antigen detection.

Method used

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  • Anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody as well as preparation method and application thereof
  • Anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody as well as preparation method and application thereof
  • Anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1: the acquisition of SARS-CoV-2 virus N protein hybridoma cell line

[0105] 1) Animal immunity

[0106] The antigen is recombinant SARS-CoV-2 virus N protein (Nanjing GenScript Biotechnology Co., Ltd., CatNo.T80103, SEQ ID NO: 1). Female Balb / c mice were subcutaneously immunized with a 1:1 emulsion containing 50 μg of SARS-CoV-2 virus N protein in 200 μl of Freund's complete adjuvant (Sigma-Aldrich). Subsequently, the mice were boosted by alternating intraperitoneal / subcutaneous injections of a 1:1 emulsion containing 25 μg of SARS-CoV-2 virus N protein in Freund's incomplete adjuvant (Sigma-Aldrich) up to 3 times every two weeks. 4 days prior to myeloma fusion, which exhibited the highest antibody titers (see figure 1 , using serum ELISA method to determine antibody titer) #1 mice were boosted intraperitoneally with 25 μg of SARS-CoV-2 virus N protein (without adjuvant).

[0107] SARS-CoV-2 virus N protein sequence (SEQ ID NO: 1):

[0108] MSDNGPQNQRNA...

Embodiment 2

[0114] Example 2: Variable region sequencing of monoclonal antibodies and antibody recombinant production

[0115]1) Use the rapid ELISA mouse antibody subtype identification kit (Clonotyping System-HRP SouthernBiotech) to identify the subtype of the antibody in the hybridoma cell culture supernatant, and the identification results show that the heavy chain is IgG1, and the light chain is Kappa type; TRIzol (Ambion) from 3×10 6 ~5×10 6 Total RNA was extracted from hybridoma cells and reverse transcribed into cDNA using antibody subtype-specific primers and universal primers (PrimeScriptTM 1stStrand cDNA Synthesis Kit, Takara).

[0116] 2) Subsequent amplification of mouse immunoglobulin heavy chain and light chain V-region fragments by RACE PCR (Nanjing KingScript Biotechnology Co., Ltd.), and subcloning the resulting PCR fragments into the pMD18-T vector system (Takara), The insert is sequenced using vector-specific primers.

[0117] 3) The unique V region amino acid seque...

Embodiment 3

[0154] Example 3: Binding of monoclonal antibodies to recombinant SARS-CoV-2 virus N protein

[0155] 1) Dilute the recombinant SARS-CoV-2 virus N protein to 0.5 μg / ml with PBS buffer.

[0156] 2) Add 100 μl of diluted SARS-CoV-2 virus N protein solution to each well of the ELISA plate (Nunc), and coat and react overnight at 4°C.

[0157] 3) Wash the plate once with PBS-T (0.05% Tween), discard the supernatant. Add 200 μl of blocking solution (100 ml of PBST; 1 g of bovine serum albumin) to each well, and incubate at 37° C. for 0.5 hour.

[0158] 4) Discard the blocking solution, add 100 μl of 10 μg / ml purified antibody to the first well, and dilute with PBS buffer according to a 3-fold gradient, a total of 7 test concentration gradients.

[0159]5) Incubate at room temperature for 1 hour. Wash the plate three times with PBST and discard the supernatant.

[0160] 6) Add 100 μl of horseradish peroxidase-labeled goat anti-human IgG (Nanjing GenScript Biotechnology Co., Ltd.)...

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Abstract

The invention belongs to the field of virus detection and diagnosis, and relates to an anti-SARS-CoV-2 virus nucleocapsid protein monoclonal antibody and a preparation method and application thereof. The invention provides the anti-SARS-CoV-2 virus nucleocapsid protein monoclonal antibody and amino acid sequences of heavy chain variable regions and light chain variable regions of the monoclonal antiboy. The anti-SARS-CoV-2 virus nucleocapsid protein monoclonal antibody provided by the invention can be specifically combined with the nucleocapsid protein. The anti-SARS-CoV-2 virus nucleocapsid protein monoclonal antibody provided by the invention provides possibility and convenience for the detection and diagnosis of the SARS-CoV-2 virus.

Description

technical field [0001] The invention belongs to the field of virus immunodetection, in particular to a monoclonal antibody against SARS-CoV-2 virus nucleocapsid protein. The present invention also relates to the preparation method of the anti-SARS-CoV-2 virus nucleocapsid protein monoclonal antibody and their application. Background technique [0002] Novel coronavirus pneumonia (COVID-19) pathogen SARS-CoV-2 virus, also known as 2019 novel coronavirus (2019 Novel coronavirus, 2019-nCoV), SARS-CoV-2 virus is a type of single-stranded positive-strand RNA virus. Its genome is about 30,000 nucleotides long. Its four main structural proteins are spike glycoprotein (S protein), small envelope protein (E protein), matrix protein (M protein) and nucleocapsid protein (N protein). The N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA. At the same time, the N protein is relatively conserved...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569
CPCC07K16/10A61P31/14G01N33/577G01N33/56983C07K2317/565C07K2317/56C07K2317/76G01N2333/165G01N2469/10
Inventor 覃喜建吴东明程朝霞
Owner NANJING GENSCRIPT BIOTECH CO LTD
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